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Molecular Cell Apr 2022Ribosome-associated quality-control (RQC) surveys incomplete nascent polypeptides produced by interrupted translation. Central players in RQC are the human ribosome- and... (Review)
Review
Ribosome-associated quality-control (RQC) surveys incomplete nascent polypeptides produced by interrupted translation. Central players in RQC are the human ribosome- and tRNA-binding protein, NEMF, and its orthologs, yeast Rqc2 and bacterial RqcH, which sense large ribosomal subunits obstructed with nascent chains and then promote nascent-chain proteolysis. In canonical eukaryotic RQC, NEMF stabilizes the LTN1/Listerin E3 ligase binding to obstructed ribosomal subunits for nascent-chain ubiquitylation. Furthermore, NEMF orthologs across evolution modify nascent chains by mediating C-terminal, untemplated polypeptide elongation. In eukaryotes, this process exposes ribosome-buried nascent-chain lysines, the ubiquitin acceptor sites, to LTN1. Remarkably, in both bacteria and eukaryotes, C-terminal tails also have an extra-ribosomal function as degrons. Here, we discuss recent findings on RQC mechanisms and briefly review how ribosomal stalling is sensed upstream of RQC, including via ribosome collisions, from an evolutionary perspective. Because RQC defects impair cellular fitness and cause neurodegeneration, this knowledge provides a framework for pathway-related biology and disease studies.
Topics: Bacteria; Humans; Peptides; Protein Biosynthesis; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 35452614
DOI: 10.1016/j.molcel.2022.03.038 -
Molecular Cell Oct 2021The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs require internal ribosome entry sites (IRES) if they are...
The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs require internal ribosome entry sites (IRES) if they are to undergo translation without a 5' cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify more than 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue-specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is downregulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease.
Topics: Cell Proliferation; Gene Expression Regulation, Neoplastic; HEK293 Cells; HeLa Cells; Humans; Internal Ribosome Entry Sites; Mutation; Neoplasms; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Circular; Receptor, Fibroblast Growth Factor, Type 1; Ribosome Subunits; Structure-Activity Relationship
PubMed: 34437836
DOI: 10.1016/j.molcel.2021.07.042 -
The EMBO Journal Apr 2023The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were... (Review)
Review
The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre-ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells. Yet, it remained a challenge for the field to assign a function to many of the identified factors and to reveal their molecular mode of action. Over the past decade, structural, biochemical, and cellular studies have largely filled this gap in knowledge and led to a detailed understanding of the molecular role that many of the players have during the stepwise process of ribosome maturation. Such detailed knowledge of the function of ribosome biogenesis factors will be key to further understand and better treat diseases linked to disturbed ribosome assembly, including ribosomopathies, as well as different types of cancer.
Topics: Humans; Ribosomal Proteins; Proteomics; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; RNA, Ribosomal
PubMed: 36762427
DOI: 10.15252/embj.2022112699 -
Molecular Cell Jul 2023Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics...
Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.
Topics: Humans; Tubulin; Ribosomes; Microtubules; Homeostasis; RNA, Messenger; RNA Stability; Carrier Proteins; Transcription Factors
PubMed: 37295431
DOI: 10.1016/j.molcel.2023.05.020 -
Molecular Cell Jun 2023Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying...
Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.
Topics: rab5 GTP-Binding Proteins; RNA, Messenger; Endosomes; Biological Transport; Endocytosis
PubMed: 37267905
DOI: 10.1016/j.molcel.2023.05.012 -
Cell May 2023Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the...
Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.
Topics: Humans; Cell Nucleolus; Eukaryotic Cells; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ribosomes
PubMed: 37172570
DOI: 10.1016/j.cell.2023.04.030 -
Genes & Development Jul 2022The nucleolus is best known for housing the highly ordered assembly line that produces ribosomal subunits. The >100 ribosome assembly factors in the nucleolus are... (Review)
Review
The nucleolus is best known for housing the highly ordered assembly line that produces ribosomal subunits. The >100 ribosome assembly factors in the nucleolus are thought to cycle between two states: an operative state (when integrated into subunit assembly intermediates) and a latent state (upon release from intermediates). Although it has become commonplace to refer to the nucleolus as "being a multilayered condensate," and this may be accurate for latent factors, there is little reason to think that such assertions pertain to the operative state of assembly factors.
Topics: Cell Nucleolus; RNA, Ribosomal
PubMed: 36342833
DOI: 10.1101/gad.349748.122 -
Nucleic Acids Research Jul 2022Human ribosomes have long been thought to be uniform factories with little regulatory function. Accumulating evidence emphasizes the heterogeneity of ribosomal protein...
Human ribosomes have long been thought to be uniform factories with little regulatory function. Accumulating evidence emphasizes the heterogeneity of ribosomal protein (RP) expression in specific cellular functions and development. However, a systematic understanding of functional relevance of RPs is lacking. Here, we surveyed translational and transcriptional changes after individual knockdown of 75 RPs, 44 from the large subunit (60S) and 31 from the small subunit (40S), by Ribo-seq and RNA-seq analyses. Deficiency of individual RPs altered specific subsets of genes transcriptionally and translationally. RP genes were under cotranslational regulation upon ribosomal stress, and deficiency of the 60S RPs and the 40S RPs had opposite effects. RP deficiency altered the expression of genes related to eight major functional classes, including the cell cycle, cellular metabolism, signal transduction and development. 60S RP deficiency led to greater inhibitory effects on cell growth than did 40S RP deficiency, through P53 signaling. Particularly, we showed that eS8/RPS8 deficiency stimulated apoptosis while eL13/RPL13 or eL18/RPL18 deficiency promoted senescence. We also validated the phenotypic impacts of uL5/RPL11 and eL15/RPL15 deficiency on retina development and angiogenesis, respectively. Overall, our study provides a valuable resource for and novel insights into ribosome regulation in cellular activities, development and diseases.
Topics: Gene Knockdown Techniques; Humans; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic; Transcription, Genetic
PubMed: 35137207
DOI: 10.1093/nar/gkac053 -
Science (New York, N.Y.) Sep 2020Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40 ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40 and various native Nsp1-40 and -80 complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
Topics: Betacoronavirus; Binding Sites; COVID-19; Coronavirus Infections; Cryoelectron Microscopy; DEAD Box Protein 58; Humans; Immune Evasion; Immunity, Innate; Interferon-beta; Models, Molecular; Pandemics; Pneumonia, Viral; Protein Binding; Protein Biosynthesis; Protein Domains; Protein Interaction Domains and Motifs; Protein Structure, Secondary; RNA, Messenger; Receptors, Immunologic; Ribosome Subunits, Small, Eukaryotic; SARS-CoV-2; Viral Nonstructural Proteins
PubMed: 32680882
DOI: 10.1126/science.abc8665 -
FEBS Open Bio Jun 2022The genome of viruses classified as picornaviruses consists of a single monocistronic positive strand RNA. The coding capacity of these RNA viruses is rather limited,... (Review)
Review
The genome of viruses classified as picornaviruses consists of a single monocistronic positive strand RNA. The coding capacity of these RNA viruses is rather limited, and thus, they rely on the cellular machinery for their viral replication cycle. Upon the entry of the virus into susceptible cells, the viral RNA initially competes with cellular mRNAs for access to the protein synthesis machinery. Not surprisingly, picornaviruses have evolved specialized strategies that successfully allow the expression of viral gene products, which we outline in this review. The main feature of all picornavirus genomes is the presence of a heavily structured RNA element on the 5´UTR, referred to as an internal ribosome entry site (IRES) element, which directs viral protein synthesis as well and, consequently, triggers the subsequent steps required for viral replication. Here, we will summarize recent studies showing that picornavirus IRES elements consist of a modular structure, providing sites of interaction for ribosome subunits, eIFs, and a selective group of RNA-binding proteins.
Topics: Internal Ribosome Entry Sites; Picornaviridae; RNA, Messenger; RNA, Viral; Virus Replication
PubMed: 35313388
DOI: 10.1002/2211-5463.13400